Development Of Genetic Manipulation For Cytophaga Hutchinsonii And Their Use

The industrial revolution has been accelerating the pace of progress of human civilization greatly, and the source of its power is the non-renewable fossil resources formed a hundred million years ago. In the past two hundred years we have been immersed in the joy it brings. Until a few decades ago. we gradually became aware of these fossil resources will soon exhausted. The same time, we still have to control environmental pollution and prevent climate continues to deteriorate. Energy supply problem is directly related to human survival and development, but also involves world peace and social stability. Therefore, to find and develop environmentally friendly renewable resources become an urgent task in countries. Solar energy is the most important source of energy for life on Earth, the biomass is immobilized solar energy. Biomass energy is abundant, there are broad prospects in bio fuel development. The most valuable part of biomass is cellulose. But there is a bottleneck problem, the cellulases’ efficiency is too low. and the cost is too expensive. An important factor leading to the low efficiency is the presence of crystalline regions in the cellulose.Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides （CFB） group which is widely distributed in earth （Walker and Warren1938; Xie et al.2007）. C. hutchinsonii could degrade crystalline cellulose rapidly and completely （Stanier1942）. However, its genome sequence does not code for any homologues of either processive cellulases, including exo-cellulases and processive endo-cellulases, or dockerin and cohesion （Wilson2009）, which are important components of cellulosome （Bayer et al.2004）. So, it utilizes an unknown strategy to degrade crystalline celluloses, which is different from both the free cellulases mechanism used by most aerobic microorganisms and the cellulosome mechanism used by most anaerobic bacteria （Wilson2009; Wilson2008）. This unknown mechanism may be helpful to improve the efficiency from crystalline cellulose into biofuel. The complete sequencing of C. hutchinsonii has provided a large amount of information （Xie et al.2007）, but functional genomics investigation have not been conducted so far because of the lack of genetic manipulation tools （Chen et al.2007b）.The only reported genetic manipulation technique for C. huichinsonii is the transposon-mediated mutagenesis by conjugation （McBride and Baker1996）. C. hutchinsonii does not have any endogenous plasmid and no broad-host-range plasmid has been reported to replicate within it. Gene targeting by suicide vectors commonly used in other CFB group organisms does not work for C. hutchinsonii due to low rate of conjugate transformation and homologous recombination. Gene manipulation has been problematic for this bacterium. Here we developed some genetic techniques and tools to study the novel cellulose degradation mechanism of C. hutchinsonii.1、This work developed replicative plasmids,carrying the replication origin of the C hutchinsonii chromosome. Plasmid is the most important tool, but we did not find any broad-host plasmid that can be used in C. hutchinsonii. Replicable plasmids have previously been constructed for bacteria lacking manipulation tools by cloning the replication origin of the chromosome (oriQ into artificial plasmids （Cordova et al.2002; Monteiro et al.2001; Ye et al.1994）. We created a replicable plasmid according to this strategy for C. hutchinsonii, and as a result, they can be used as Escherichia coli-C. hutchinsonii shuttle vectors. Then there transformation efficiency and stability were compared to choose for different purposes. Such as plasmid containing the standard length of replication origin fragment has lower efficiency, but its probability of integration into the genome is also low, so it would be more suitable for the knockout. 2、Using the replicable plasmid, we developed a method for electro-transformation. Before this work, the only reported genetic manipulation technique for C. hutchinsonii is transposon-mediated mutagenesis by conjugation with Escherichia coli. Finally a transformation efficiency of about2×103transformants per microgram plasmid DNA or transformation frenquency of10-5as achieved. Higher transformation efficiency can be compensate for the low efficiency of the C. hutchinsonii homologous recombination defects.3、Some heterologous genes, including green fluorescent protein, galactose kinase and β-galactosidase. were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU₁284promoter in oriC plasmids. Studies on expression regulatory elements in Bacteroides is rare. Some reporter genes cannot be used in C. hutchinsonii directly. The expression elements such as the promoter must be modified in C. hutchinsonii. promoters in C. hutchinsonii are found very similar to Flavobacterium hibernum, they do not have the-35and-10conserved sequence, but-33and-7. The ribosome binding sequence in C. hutchinsonii is similar to Flavobacterium hibernum.4、A comparative proteomic analysis of C. hutchinsonii was performed, and found some interesting proteins maybe taking part in cellulose degradation. We performed a comparative proteomic analysis of the total cell proteins of C. hutchinsonii cultured in Stanier’s mineral base medium with either glucose or filter paper as the sole carbon source. The significantly up-regulated proteins induced by cellulose were identified by MS. The up-regulated proteins were mainly related to oxidative stress response and amino acid metabolism. The cells were under oxidative stress and might be in a kind of biofilm when they growing on cellulose fiber.5、The replicative plasmid was successfully used for gene targeting. The homologous recombination efficiency in C. hutchinsonii is very low, so, gene targeting by ordinary suicide plasmid is very difficult. In this work, we developed an efficient knockout method, it is based on the oriC plasmid with two-step screening. The first step, screen of transformants having erythromycin resistance. Second step, the screen out chloramphenicol resistance colonies. The cat gene on the plasmid does not have its own promoter, so transformant only having free plasmid does not has chloramphenicol resistance. After homologous recombination, the plasmid integrates into the target gene, and the cat gene can be transcript by promoter upstream of target gene. These genetic tools will be useful in functional genomics investigations of the cellulolytic6、Some genes were knocked out and some phenotypes of the mutants were investigated. Some genes, specially related to gliding and porSS, were selected for gene targeting. C. hutchinsonii does not have cell surface flagella or other similar motion organs, but could move fast on the wet surface, just like Flavobaclerium johnsoniae. Bio informatics analysis shows that C. hutchinsonii has a full set of gliding related proteins. The cellulose degradation of C. hutchinsonii was thought to be related to gliding motility before. But the phenotypes studies of these mutants reveals the relationship between cellulose degradation and spreading is not so close.We believe these tools will be useful in the investigation of C. hutchinsonii.